Doctoral Degrees (Physiological Sciences)

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Browsing Doctoral Degrees (Physiological Sciences) by Author "Goldswain, Toni Leigh"

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    An investigation into the potential cardioprotective effects of ghrelin in a rat model of chronic Doxorubicin-induced cardiotoxicity
    (Stellenbosch : Stellenbosch University, 2017-12) Goldswain, Toni Leigh; Sishi, Balindiwe J. N.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH SUMMARY: Introduction Doxorubicin (DOX) is an anthracycline that has significantly improved the outcome of cancer patients since its discovery. However, its clinical success remains limited due to the dose-dependent cardiotoxic side effects associated with its use. While the mechanisms responsible for this condition are still not well defined, oxidative stress alongside apoptosis, remain the classical but major contributors. Ghrelin, an endogenous brain-gut peptide, most well-known for its effects on appetite and growth hormone release, has been shown to exert anti-apoptotic, antioxidative, anti-inflammatory and anti-fibrotic effects in several models of cardiovascular disease. These cardioprotective effects offered by ghrelin have been indicated to occur through the activation of the pro-survival proteins, ERK1/2, Akt/PKB and STAT3. This study therefore investigated the protective effects of ghrelin in a chronic model of DOXinduced cardiotoxicity. Methods Male Sprague-Dawley rats were acclimatized and divided into five experimental groups. While the control group remained untreated, the ghrelin group received 300 μg/kg ghrelin per week, the DOX group received 2.5 mg/kg per week, the combination group received both DOX and ghrelin at the previously mentioned doses and the vehicle group received saline. All injections were performed via intraperitoneal injection for eight weeks. One week after the last injection, the rats were euthanized, blood was collected and the hearts were subjected to a 40-minute working heart perfusion protocol to obtain functional data. The hearts were weighed and then cut transversely into two sections, where one half was snap frozen for biochemical analysis and the other half was preserved in formalin for histological analysis. Cardiovascular markers of damage and pro-inflammatory cytokines were measured in serum using a luminex assay, and fibrosis and general morphology were assessed using the Masson’s Trichrome and H&E stains, respectively. Cytochrome c expression was evaluated by immunohistochemistry, while oxidative stress was assessed using the TBARS, conjugated diene, ORAC, SOD and glutathione assays. Apoptosis was determined using the Caspase Glo® assay and the expression of pro-survival proteins was measured using Western blot analysis. Results During the eight weeks of treatment, DOX significantly reduced weight gain and food consumption, whereas ghrelin maintained normal body weight and stimulated appetite. As expected, DOX induced significant oxidative stress when compared to the control, as demonstrated by the formation of conjugated dienes (1.64 ± 0.11 vs 0.55 ± 0.12 μmol/gram, p = 0.0003) and a reduction in the GSH:GSSG ratio (2.10 ± 0.47 vs 9.66 ± 1.08 arbitrary units, p< 0.05), whereas ghrelin attenuated these effects. Apoptotic cell death was also induced, as evident by an increase in cytochrome c staining, caspase activity (67877 ± 15686 vs 13421 ± 1871 relative light units, p< 0.0001) and PARP cleavage (2.11 ± 0.24 vs 1.00 ± 0.09 fold change, p = 0.0081). The decrease in cell death and oxidative stress in this scenario was associated with a reduction in TNF- and an improvement in cardiac function, which was otherwise worsened in the DOX group due to the decline cardiac output, coronary flow, ratepressure product, left ventricular developed pressure and total work. Even though ghrelin treatment in the presence of DOX did not induce noteworthy changes in the protein expression of ERK1/2 and Akt/PKB, the phosphorylation of STAT3 was improved with ghrelin administration. Discussion and Conclusion The observations presented in this study indicate that while DOX is a known effective chemotherapeutic agent, it produces cardiotoxic effects through the induction of oxidative stress and consequently apoptosis, possibly due to the downregulation of ERK1/2 and Akt/PKB. The co-administration of ghrelin with DOX significantly decreased the detrimental effects associated with DOX treatment alone. The effects of ghrelin were not only beneficial at organ level, but also at the organism level, as a result of improved general well-being of the experimental animals and through the maintenance of cardiac function, a decline in myocardial TNF- production and the stimulation of the STAT3 signaling pathway. The fact that ghrelin alone did not exert any detrimental effects makes this peptide an appealing cardioprotective agent and may therefore have the potential to improve the high morbidity and mortality rates of cancer survivors. While ghrelin in this context may possess anticardiotoxic effects, further research is required to determine the effects of ghrelin on cancer cell proliferation.